Biologically active substances such as nucleic acid, antibody, and antigen existing in a specimen are detected and identified in certain fields such as clinical examination, food inspection, and medicolegal inspection. Several methods are used for such detection and identification depending on objective substances, including, for example, nucleic acid probe techniques and enzyme immunoassay methods.
The field, in which nucleic acid is detected, includes, for example, identification of microbial species such as pathogenic microorganisms, and DNA tests in legal medicine. Usually, in the detection of nucleic acid, a nucleic acid having a sequence complementary to a target nucleic acid is used, which is directly labeled with an enzyme or the like, or indirectly labeled through a hapten or the like. The labeled nucleic acid is hybridized with the target nucleic acid. The presence and the amount of the target nucleic acid can be confirmed by detecting a hybridized labeled portion after removing a non-hybridized part of the labeled nucleic acid or after inactivating a non-hybridized labeled portion.
The field, in which antigen, antibody or the like is detected, includes, for example, identification of microbial species such as pathogenic microorganisms in the same manner as described for the nucleic acid, as well as various clinical examinations. A competitive enzyme immunoassay method, which is an embodiment of the enzyme immunoassay method used to detect antigen and antibody, is performed as follows. An antibody or an antigen is immobilized on a surface of a solid phase such as polystyrene bead, microtiter plate, and tube. A certain amount of a specimen solution is applied to the solid phase surface, and then an antigen-enzyme complex or an antibody-enzyme complex is added. When the antibody is immobilized on the solid phase surface, the antigen in the specimen and the antigen-enzyme complex compete in a binding reaction to the antibody immobilized on the solid phase surface. When the antigen is immobilized on the solid phase surface, the antigen in the specimen and the antigen immobilized on the solid phase surface compete in a binding reaction to the antibody-enzyme complex.
After passage of a certain period of time, when the antibody is immobilized, the antigen and the antigen-enzyme complex, which do not bind to the immobilized antibody, are washed and removed. When the antigen is immobilized, unreacted parts of the antigen in the specimen and the antibody-enzyme complex, and a bound product between the antigen i n the specimen and the antibody-enzyme complex are washed and removed. The washing in this procedure is usually performed by repeating a washing operation several times to ten times. The washing operation comprises filling a solid phase section with a washing solution, discarding the washing solution, filling the solid phase section with a fresh washing solution again, and discarding the washing solution again. This operation is generally called "B/F separation" which is an essential operation in the inspection based on the principle of the enzyme immunoassay method.
In the final step, a coloring substrate solution for the enzyme used to label the antigen or the antibody is added to the solid phase section, and then a color is developed by the remaining enzyme. Generally, the enzyme used in this procedure is, for example, peroxidase, .beta.-galactosidase, and alkaline phosphatase. A substrate appropriate for each enzyme is used as the substrate for color development. If the antigen as the target exists in a large amount in the specimen solution, the amount of the remaining antigen-enzyme complex or the remaining antibody-enzyme complex is decreased, resulting in a weak intensity of color development. The intensity of color development is generally measured by using a calorimeter.
In a sandwich enzyme immunoassay method as another embodiment of the enzyme immunoassay method, an antibody is immobilized on a solid phase surface, and a specimen solution is applied to the solid phase surface. After passage of a certain period of time, a part of an antigen which does not bind to the antibody on the solid phase surface is washed and removed. After that, a certain amount of an antibody-enzyme complex is added. After passage of a certain period of time, a part of the antibody-enzyme complex which does not bind to the antigen on the solid phase surface is washed and removed, and then a coloring substrate is applied to the solid phase surface to develop a color. The concentration of the antigen in the specimen can be quantitatively determined by measuring the intensity of the color development.
It is extremely important to immobilize antibody, antigen, enzyme, nucleic acid, or the like on the solid phase surface such as tube, microtiter plate, membrane filter, and beads in the conventional methods such as the nucleic acid detection method, the competitive enzyme immunoassay method, and the sandwich enzyme immunoassay method described above. Accordingly, various methods for immobilizing biologically active substances have been published. For example, known methods for protein include:
(1) a method for chemically binding a protein to a base material by using a cross-linking agent or a condensation agent, such as a diazo method, a peptide method, an alkylation method, a base material-binding method by using a cross-linking agent, and a base material-binding method based on Ugi reaction (see "Immobilized Enzyme", ed. by Ichiro Chibata, Kodansha Scientific (1986), pp. 9-41); PA1 (2) a method for immobilization to a base material by using ionic bond (see "Immobilized Enzyme", pp. 41-43); and PA1 (3) a method for immobilization to a base material by using physical adsorption (see "Immobilized Enzyme", pp. 43-45). PA1 (1) a method for chemically binding nucleic acid which is provided with an introduced modified group, as exemplified by immobilization through a disulfide bond between nucleic acid having a thiol group at its 5'-terminal and a bead-shaped base material involving a thiol group (see P. J. R. Day, P. S. Flora, J. E. Fox, M. R. Walker, Biochm. J., 278, 735-740 (1991)) (other methods belonging to this category are described in, for example, Soren R. R., Mette R. L., Svend E. R., Anal. Biochm., 198, 138-142 (1991); Jonathan N. K., Joseph L. W., Joseph P. D., Rachel E. M., Mary C., Eugene L. B., Nucleic Acids Res., 15, 2891-2909 (1987); Allan J. M., Jeffrey R. B., Terence W. P., Biochem. J., 191, 855-858 (1980); J. A. Running, M. S. Urdea, BioTechniques, 8, 276-279 (1990)); and PA1 (2) a method for immobilizing nucleic acid by using physical adsorption such as immobilization by adsorption onto a nitrocellulose or nylon membrane by means of UV irradiation or heat treatment (J. Sambrok, E. F. Fritsch and T. Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory Press, Second Edition, pages 2.109-2.113 and pages 9.34-9.46), and immobilization by physical adsorption onto a microplate (G. C. N. Parry and A. D. B. Malcolm, Biochem. Soc. Trans., 17, 230-231 (1989). PA1 reacting a biologically active first substance immobilized on a carrier with a second substance capable of specifically binding the first substance; and PA1 detecting a non-bound part of the second substance or a bound part of the second substance indirectly bound to the carrier through binding between the first and second substances so that the first substance or the second substance in a sample is analyzed; PA1 wherein the carrier comprises a compound having 2 to 100 carbodiimide groups, and the first substance is immobilized on the carrier through the carbodiimide groups.
Known methods for nucleic acid include, for example:
However, it has been pointed out that the conventional methods as described above have drawbacks. For example, the method based on chemical bond requires special reagents including, for example, toxic substances such as azide, isocyanate, and NaBH.sub.3 CN. Further, when immobilization is performed by using peptide bond, for example, it is necessary to introduce an amino group into any one of an active substance and a base material, and it is necessary to introduce a carboxyl group into the other. Moreover, it is necessary to provide a step of mutually treating both of the introduced functional groups with a condensation reagent to achieve immobilization. Accordingly, it is inevitable to suffer complicated operations.
In the case of the method based on chemical bond, for example, amino groups must be present in both of the base material and the active substance in order to use glutaraldehyde as a cross-linking agent. Accordingly, it is necessary to select the base material because the base material itself must have the functional group. As a result, it becomes difficult to select the base material suitable for immobilization. In addition, for example, the method base on a chemical reaction is difficult to be used for those having only functional groups with poor reactivity (for example, terminal phosphate group, and terminal hydroxyl group) such as natural DNA and DNA having no modified group. Thus the method based on chemical bond has a drawback that immobilization cannot be achieved when the active substance has no active functional group.
On the other hand, the physical adsorption has the following drawback. Namely, the amount of immobilization is affected by adsorption performance of the base material, and the adsorbed active substance is liable to desorb. When the active substance is a low-molecular weight compound (oligomer), it is scarcely adsorbed because it has weak interaction with the base material.
As described above, many problems remain in the immobilization which is important to detect active substances such as protein and nucleic acid.